Estrogenic compounds and animal growth promoters



United States Patent Ofi ice 3,239,341 ESTROGENIC COMPOUNDS AND ANIMALGROWTH PROMOTERS Edward B. Hodge, Phil H. Hidy, and Herbert L.Wehrmeister, Terre Haute, Ind., assignors to Commercial SolventsCorporation, a corporation of Maryland No Drawing. Filed Feb. 15, 1965,Ser. No. 432,811 20 Claims. (Cl. 99-2) The present invention relates tonew compounds and an object of the present invention is to providecompounds which exhibit estrogenic activity or aid in increasing therate of growth in meat-producing animals, e.g. cattle, lamb and swine.

A conventional formula for the compounds of the present invention is:

(CHM

where A is --CH CH or CH=OH; R is hydro gen, substituted orunsubstituted alkyl, e.g. lower alkyl such as methyl, ethyl, hexyl,etc., and acyl, e.g. lower saturated acyclic acyl radicals such asacetyl andvaleryl, but hydrogen is preferred. Compounds having the aboveformula where R is substituted or unsubstituted aryl, e.g. phenyl andbromophenyl, and aralkyl, e.g. benzyl, are also contemplated by thepresent invention.

The compounds can be administered to animals by any suitable methodincluding oral and parenteral administrations. For example, the compoundcan be blended with ordinary feed containing nutritional values in anamount sufiicient to .produce the desired rate of growth and can thus befed directly to the animals, or the compound can be suspended in asuitable injection suspension medium such as peanut oil and injectedparenterally. The amount of compound fed to an animal, of course, variesdepending upon the animal, desired .rate of growth and the like.

When the new compounds are to be administered in feeds, an animal feedcomposition may be prepared containing the usual nutritionally-balancedquantities of carbohydrates, proteins, vitamins and minerals, togetherwith the compound of the present invention. Some of these usual dietaryelements are grains, such as ground grain and grain by-pr-oducts; animalprotein substances, such as those found in fish meal and meat scraps;vegetable proteins like soybean oil meal or peanut oil meal;vitaminaceous materials, e.g. vitamin A and D mixtures; riboilavingsupplements and other vitamin B complex members; and bone meal andlimestone to provide minerals. A type of conventional feed material foruse with cattle includes alfalfa hay and ground corn cobs together withsupplementary vitaminaceous substances if desired.

The compounds of the present invention can be produced from thecompound:

hereinafter referred to as the fermentation estrogenic substance(F.E.S.) by reduction of the ketone group to replace the oxygen of theketone group with two hydrogen atoms. The reduction of the ketone groupcan be effected by several procedures. One of these procedures involvesthe C-lemmensen reduction using zinc and hydrochloric acid; anotherinvolves the Woltf-Kishner re- 3 ,239,34 Patented Mar. 8, 1966 ductionusing hydrazine and alkali, e.g. NaOH, and the third involves formationof the dithioacetal with ethylene dithiol or ethylmercaptan and thecatalytic desulfurization with Raney nickel catalyst containing adsorbedhydrogen.

In producnig compounds of the present invention where A is -CH CH theolefinic bond of F.E.S. can be reduced, for example, by hydrogenation inthe presence of a Group VIII metal, particularly platinum or palladiumcatalyst on a suitable carrier, e.g. charcoal. Generally, the catalystcontains from about 0.01 to about 10% of the catalytic metal. Thecatalyst is used in a ratio of generally from 0.02 to 2 grams ofcatalyst, preferably about 0.1 to 0.5 gram, and particularly about 0.2gram catalyst per gram of F.E.S. The reduction may be carried out whilethe F.E.S. is dissolved in a suitable solvent, e.g. an alcohol,especially a lower alkanol such as 2-propanol, methanol, ethanol, andacid, e.g. acetic acid, etc. at ambient temperatures; e.g. from about 15to 40 C., and ambient pressures, since only the presence of hydrogen isrequired; however, it is preferred to utilize an elevated pressure, e.g.from about 1 to S0 atmospheres of hydrogen.

In producing compounds of the present invention where R is alkyl,conventional alkylation procedures can be used to replace the H atom ofone or both of the OH groups on the benzene ring of F.E.S. with an alkylgroup. Alkyl-ated dihydro F.E.S. compounds can be produced, for example,by first alkylating F.E.S. and then reducing it as set forth supra, orby first reducing it and then alkylating it. The alkylation can be byreaction with the corresponding dialkyl sulfates, e.g. dimethyl sulfate,diethyl sulfate, etc., to produce the dialkyl F.E.S. or a monoalkylF.E.S. with the alkyl group replacing the hydrogen of the hydroxyl groupon the benzene ring ortho to the ester group. Furthermore, a monomethylcompound with the methyl group replacing the hydrogen of the hydroxylgroup para to the ester group can be selectively produced usingdiazomethane.

In producing compounds of the present invention where R is acyl,conventional acylation procedures can be used to replace the hydrogenatom of one or both of the hydroxyl radicals on the benzene ring ofF.E.S. with an acyl radical. Acylated F.E.S. compounds can be produced,for example, by reaction with the corresponding acid anhydride, e.g.acetic anhydride, propionic anhydride, etc., catalyzed with, forexample, sodium acetate or pyridine. Ambient conditions can be usedalthough it is preferred to keep the reaction mixture cold. Whencompounds having one R as alkyl and the other acyl, it is advantageousto alkylate before acylating.

The fermentation estrogenic substance (F.E.S.) is so named since aconvenient method for producing it is by cultivating, on a suitablenutrient medium, the organism Gibberella zeae (Gordon) on deposit at theNorthern Utilization Research and Development Division of the UnitedStates Department of Agriculture under the number NRRL-2830.

The following examples are offered to illustrate this invention;however, the invention is not limited to the specific materials,amounts, and procedures set forth. The first example illustratespreparation of a suitable inoculum containing the organism Gibberellazeae (Gordon) NRRL- 2830.

Example I A spore sand culture containing Gibberella zeae (Gordon)NRRL-2830 was aseptically placed in a sterile tube containing 15milliliters of Czapeks-Dox solution and a small amount of agar. Thismedium was then incubated for about 168 hours at approximately 25 C. Atthe end of the incubation period, the medium was washed with 5milliliters of sterile deionized water and transferred to a sterile tubecontaining 45 milliliters of Czapeks-Dox solution. The contents of thetube were then incubated for about 96 hours at about 25 C. after whichthe material was available for use in inoculation of a fermentationmedium.

The following example illustrates the fermentation of the organismGibberella zeae (Gordon) NRRL-2830 to produces F.E.S.

Example II To a 2 liter flask were added 300 grams of finely dividedcorn. The flask and its contents were then sterilized and aftersterilization 150 milliliters of sterile deionized water were added. Tothe mixture in the flask were then added 45 milliliters of the inoculumprepared by the process of Example I and the material was thoroughlymixed. The mixed material was then incubated for about 20 days at 25 C.in a dark room in a water-saturated atmosphere.

The following example illustrates the recovery of F.E.S. from thefermentation medium.

Example III A 300 gram portion of fermented material produced by themethod of Example II was placed in 500 milliliters of deionized waterand slurried. The slurry was then heated for about 15 minutes at 75 C.,300 grams of filter aid were then added and the material was filtered.The solid filtered material containing the anabolic substance was thenair dried, and 333 grams of the dried cake were then extracted with 500milliliters of ethanol. This procedure was repeated three more times.The ethanol extract was evaporated to dryness under vacuum to give 6.84grams of solid material. This solid material was then dissolved in 20milliliters of chloroform and extracted with 30 milliliters of anaqueous solution containing 5% by weight of sodium carbonate having anadjusted pH of about 11.2. The extraction process was repeated sevenmore times. The pH of the sodium-carbonate extract was then adjusted to6.2 with hydrochloric acid, to yield an anabolic substance-containingprecipitate. The precipitate and the aqueous sodium carbonate extractwere then each in turn extracted with 75 milliliters of ethyl ether.This procedure was repeated three more times to yield a light yellowethereal solution, which was then evaporated to yield 116 milligrams ofsolid anabolic substance. This material was then subjected to multipletransfer countercurrent distribution using 100 tubes and a solventsystem consisting of two parts chloroform and two parts carbontetrachloride as the lower phase and four parts methanol and one partwater as the upper phase, all parts by volume. The solid materialobtained from the multiple transfer countercurrent distribution wasF.E.S.

Example IV Two gram portions of F.E.S., each in 200 milliliters aceticacid, were catalytically reduced at room temperature in the presence of1.2 grams of PdO catalyst at a hydrogen pressure of about 45 p.s.i. Thecombined reduction mixtures were heated to boiling, filtered, and thefilter cake was washed with 50 milliliters of hot acetic acid. Thecooled filtrate was added, with stirring, to 2 liters of water. Themixture was stirred for minutes and the white solid was collected byfiltration, washed and dried in a vacuum desiccator to yield 19.1 gramsof dihydro F.E.S. having a melting point of 191193 C.

The dihydro F.E.S. (1 gram) is added slowly with cooling (ice-bath), toa mixture of 5 cc. of ethylene dithiol .25 gram of freshly fused zincchloride and 2 grams of anhydrous sodium sulfate, contained in amicroflask. The mixture is maintained at 5 C. for hours and then at roomtemperature for 4 hours, whereupon it is poured into 50 cc. of ice andthe precipitate is collected and subjected to hydrogenolysis. To thereaction product is added 100 cc. of 90% ethanol and 15 grams of Raneynickel catalyst and the mixture is refluxed until the reaction iscomplete. The nickel is removed by centrifugation and is washed severaltimes with hot ethanol by centrifugation followed by decantation, andthe centrifugates are combined. The mixture is evaporated to dryness andthe residue is suitably recrystallized to yield a compound having theformula:

H0 o1n).

Example V The procedure used in Example IV is essentially followed withthe exception that F.E.S. is used instead of dihydro F.E.S. to produce acompound having the formula:

The following example illustrates the production of a monomethyl F.E.S.derivative having the methyl group replacing the hydrogen in thehydroxyl group para to the ester group of F.E.S.

Example VI Nitrosomethylurea in an amount of 1.2 grams was slowly addedto a cold mixture of 3.6 milliliters of 50% potassium hydroxide and 17milliliters of ether. After a few minutes the yellow ether layer of themixture was decanted, dried over potassium hydroxide, and then added toa solution of 0.30 gram F.E.S. in 17 milliliters of ether. The resultingyellow mixture was left overnight in a loosely stoppered flask and thenether and diazomethane were evaporated using a steam bath. The remaininggummy residue was crystallized by adding 3 milliliters of water, heatingto 60 C., and adding ethanol almost to solution. On cooling, crystalsformed yielding 0.137 gram of a product having a melting point of111-116 C. which was recrystallized in the same way to yield 0.082 gramof monomethyl F.E.S. having a melting point of The p-methyl F.E.S. issubstituted for the dihydro F.E.S. in following essentially the sameprocedure used in Example IV to produce a compound having the formula:

HO (I? CH3 The following example illustrates the production of dimethylF.E.S. and monomethyl F.E.S. derivatives, the monomethyl F.E.S.derivative having the hydrogen in the hydroxyl group ortho to the estergroup replaced with a methyl group.

5 Example VII Dimethyl sulfate (5 milliliters) was added to a solution 6Example IX The compound:

of 2.24 grams of PBS. in 80 milliliters 10% NaOH and OHPO 20 milliliterswater. The mixture was stirred for onei half hour at 1820 C. (coolingbath) and an additional 5 5 milliliters of dimethyl sulfate was added.After an additional 70 minutes of stirring at 2026 C., the solid c -c 0precipitate, Solid A, was collected by filtration, washed GH=CH-(OHi)awith water and dried in a vacuum desiccator. The filr trate from Solid Awas acidified with 25 milliliters 12 N g gfg g g 32 2 5322 1 g gg gg jif the H2504 Yield a Precimtat? S1id whlch was The roduction of thedimethyldihydi' o F.E .S. derivaconecied washed with i and dnedi tive isillustrated by the following example.

Solid A (0.79 gram having a melting point of 114 118 C.) wasrecrystallized from a mixture- 0f 10 milli- E l X liters water and 15milliliters ethanol to yield 0.66 gram o Dlhydro RES. (556 milligrams)was dissolved 1n fi i g havmg a meltmgypomt 108 milliliters 10% NaOH and1d milliliters water and o the solution was stirred. To the stirredsolution was I 58 S gzixi figgg f g ggi g gi lg added three,two-milliliter portions of dimethyl sulfate at water alcohol t s eofgmonomethyl 2 5..i i?fiiiiie lili ifidi a ii ii wii ifiidi iiiiifie zafneltmg 169 T and tha by the addition of 10 milliliters 10% NaOH andthe followmg analysis of recrystanlzed Sohd B (monomethyl alkalinemixture was stirred for one-half hour. The solid was obtamed' formed wascollected by filtration, washed with water 5 and dried in a vacuumdesiccator. The product weighed Cale. Found 526 milligrams and melted at115117 C. Recrystal- (CWHHOQ lization from a mixture of 10 millilitersof water and 0 7 7 25 milliliters of ethanol provided 371 milligrams ofma- Pement 68-55 6 terial having a melting point of 124125.5 C. It was.16 1 3223 gm, 5.3% 3.28 analyzed with the following results:

Cale. Found Each of the o-methyl F.E.S. and the dimethyl F.E.S.(C20H28O5) is substituted for the dihydro RES. in the procedure ofExample IV to produce the respective compounds: ercen C 68.95 69. 02Percent H 8. l0 8. 12 CHaO a (EH3 Percent C1130 17. 81 17.81

CO-CH(CHz)z The dimethyldihydro F.E.S. is substituted for dihydro CH2RES. in the procedure set forth in Example 1V to pro- HO duce a compoundhaving the formula:

CH:CH-(CH2)3 CH3-() 0 CH and H C-OCH(CH2)a c1130 6 one I /CO-CH(CH2)aoHio (oHni CH2 Example XI CH3O Six head of cattle are fed a daily rationincluding a 2 a mixture of alfalfa hay and ground corn cobs containingThe following example illustrates the production of from 1 to 20 Ouncesof the p l Produced in EXaman acylated h 1F E s,d i i ple V per hundredpounds of ration to increase the rate E 1 VI of growth of the cattle. Tl t' f ass ii' t thyl FES Example o a so u ion 0 m1 lgrams o p-me 1n 8milliliters pyridine is added 5 milliliters acetic ani f g gg fi f i g si g g gg g fi gi lfiggi fi i hydnde and the mixture i Fi at roomtemperature for from 1 to 20 ounces o l the compound produced in Exam 16i Twenty'five mllhhteis of Water are then added ple X per hundred poundsof ration to increase the rate The mixture is stored in a refrigeratorfor 2 hours. The of rowth of the cattle solid precipitated is collectedby filtration, Washed with is claimed. water and dried in a vacuumdesiccator to recover a 1 compound which is substituted for the dihydroRES. in 0 CH the procedure of Example IV to produce a compound of R O ga the formula: -O-CH(CH 0 CH3 R O oHioo i l-o-t nrrolii A (CHM where Ais selected from the group consisting of CH: CHFO CHCH-- and CH CH and Ris selected from the group consisting of hydrogen, lower alkyl and lowersaturated acyclic acyl.

2. The compound of claim 1 wherein A is CH=CH 3. The compound of claim 2wherein R is hydrogen.

4. An animal feed comprising a nutritional diluent and growth promotingamounts of the compound of claim 1.

5. The compound of claim 2 wherein R is methyl.

6. The compound of claim 2 wherein the R ortho to the ester group ismethyl and the other R is hydrogen.

7. The compound of claim 2 wherein the R para to the ester group ismethyl and the other R is hydrogen.

8. The compound of claim 2 wherein the R ortho to the ester group ismethyl and the other R is acetyl.

9. The compound of claim 2 wherein the R para to the ester group ismethyl and the other R is acetyl.

10. The compound of claim 1 wherein A .is

11. The compound of claim 10 wherein R is hydrogen. 12. The compound ofclaim 10 wherein R is methyl. 13. An animal feed comprising anutritional diluent and growth promoting amounts of the compound ofclaim 3. 14. An animal feed comprising a nutritional diluent and growthpromoting amounts of the compound of claim 5. 15. An animal feedcomprising a nutritional diluent and growth promoting amounts of thecompound of claim 6. 16. An animal feed comprising a nutritional diluentand 5 growth promoting amounts of the compound of claim 7. 17. An animalfeed comprising a nutritional diluent and growth promoting amounts ofthe compound of claim 8. 18. An animal feed comprising a nutritionaldiluent and growth promoting amounts of the compound of claim 9. 19. Ananimal feed comprising a nutritional diluent and growth promotingamounts of the compound of claim 11. 20. An animal feed comprising anutritional diluent and growth promoting amounts of the compound ofclaim 12.

A. LOUIS MONACELL, Primary Examiner.

4. AN ANIMAL FEED COMPRISING A NUTRITIONAL DILUENT AND GROWTH PROMOTINGAMOUNTS OF THE COMPOUND OF CLAIM 1.